Artemisia annua L. (sweet wormwood or artemisia), the only commercial source of the antimalarial artemisinin (ART), has genotypes with a wide range of ART concentrations and varying blooming periods. This makes the cross-pollination of high-ART genotypes difficult unless the flowering of selected parents is synchronized. In this study, we evaluated the phenological pattern of artemisia to establish a phenological scale that will allow successful cross-pollination and breeding. The scale was based on daily observations and photographs of the plant at specific time intervals and was divided into two distinct phases: the vegetative phase (with five stages) and the reproductive phase (with 12 stages). Although short days induce flowering, our experiments with Chinese and Vietnamese genotypes of artemisia demonstrated that genotypes from different geographic origins are better synchronized on both short days and low temperatures, with the Chinese genotype requiring only short days to flower and the Vietnamese genotype requiring short days and low/moderate temperatures to reach full flowering. We also noticed that Chinese plants flowering under short days can be reversed to the vegetative stage if plants are exposed to long photoperiod early enough. Finally, plants growing under long days, in the vegetative phase, have higher densities of glandular trichomes and higher concentrations of ART when compared to plants grown under short days and induced to flower. These observations suggest that the further from the inductive photoperiod the transplantation to the field occurs, the higher the concentration and yield of ART.

Abstract We assessed bioactivity of ethanolic extracts from 35 species of Jatropha L. against an ornamental plant pest, the azalea lace bug, Stephanitis pyrioides (Scott). Jatropha extracts were prepared by air-drying stem, root, or whole plant material, grinding the tissue into a fine powder, adding 70% ethanol, and then vacuum filtering the contents. Emulsions included the extract diluted to the desired concentration in de-ionized water and 10% dimethyl sulfoxide (DMSO). Treatments involved pipetting 20 µl of emulsion onto three adult lace bugs in each well of a 96-well microtiter plate. Treated wells served as replicates for each of six extract concentrations and were arranged according to a RCBD. Extracts of Jatropha clavuligera Müll. Arg. and J. ribifolia (Pohl) Ballion from 0.06 to 0.50% were the most acutely bioactive with bug mortality exceeding that of the positive control – azadirachtin, a terpenoid and chief active ingredient in neem oil. At 1.00%, extracts of J. clavuligera, J. ribifolia and azadirachtin killed 100% of bugs within 3 hr. Jatropha clavuligera induced the lowest LC50 and ranked first in insecticidal potency based on ≥98% of bugs dying within 3 hr. Extracts of J. curcas L., J. gossypiifolia L., J. excisa Griseb, and azadirachtin were equally bioactive; although after 3 hr, the three Jatropha species killed bugs faster. When compared with DMSO, all extract emulsions were bioactive against adult bugs. Thus, active ingredients in a new biopesticide could be sourced from the stem, root, or whole plant extracts of at least five Jatropha species.

We previously demonstrated that extracts from Echinacea purpurea material varied substantially in their ability to activate macrophages in vitro and that this variation was due to differences in their content of bacterial components. The purpose of the current study was to identify soil conditions (organic matter, nitrogen, and moisture content) that alter the macrophage activation potential of E. purpurea and determine whether these changes in activity correspond to shifts in the plant-associated microbiome. Increased levels of soil organic matter significantly enhanced macrophage activation exhibited by the root extracts of E. purpurea (p < 0.0001). A change in soil organic matter content from 5.6% to 67.4% led to a 4.2-fold increase in the macrophage activation potential of extracts from E. purpurea. Bacterial communities also differed significantly between root materials cultivated in soils with different levels of organic matter (p < 0.001). These results indicate that the level of soil organic matter is an agricultural factor that can alter the bacterial microbiome, and thereby the activity, of E. purpurea roots. Since ingestion of bacterial preparation (e.g., probiotics) is reported to impact human health, it is likely that the medicinal value of Echinacea is influenced by cultivation conditions that alter its associated bacterial community.

The endophytic fungal community associated with the ethnomedicinal plant Echinacea purpurea was investigated as well as its potential for providing antifungal compounds against plant pathogenic fungi. A total of 233 endophytic fungal isolates were obtained and classified into 42 different taxa of 16 genera, of which Alternaria alternata, Colletotrichum dematium, and Stagonosporopsis sp. 2 are the most frequent colonizers. The extracts of 29 endophytic fungi displayed activities against important phytopathogenic fungi. Eight antifungal extracts were selected for chemical analysis. Forty fatty acids were identified by gas chromatography-flame-ionization detection (GC-FID) analysis. The compounds (-)-5-methylmellein and (-)(3R)-8-hydroxy-6-methoxy-3,5-dimethyl-3,4-dihydroisocoumarin were isolated from Biscogniauxia mediterranea EPU38CA crude extract. (-)-5-Methylmellein showed weak activity against Phomopsis obscurans, P. viticola, and Fusarium oxysporum, and caused growth stimulation of C. fragariae, C. acutatum, C. gloeosporioides, and Botrytis cinerea. (-)-(3R)-8-Hydroxy-6-methoxy-3,5-dimethyl-3,4-dihydroisocoumarin appeared slightly more active in the microtiter environment than 5-methylmellein. Our results indicate that E. purpurea lives symbiotically with different endophytic fungi, which are able to produce bioactive fatty acids and aromatic compounds active against important phytopathogenic fungi. The detection of the different fatty acids and aromatic compounds produced by the endophytic community associated with wild E. purpurea suggests that it may have intrinsic mutualistic resistance against phytopathogen attacks in its natural environment.

Evidence supports the theory that bacterial communities colonizing Echinacea purpurea contribute to the innate immune enhancing activity of this botanical. Previously, we reported that only about half of the variation in in vitro monocyte stimulating activity exhibited by E. purpurea extracts could be accounted for by total bacterial load within the plant material. In the current study, we test the hypothesis that the type of bacteria, in addition to bacterial load, is necessary to fully account for extract activity. Bacterial community composition within commercial and freshly harvested (wild and cultivated) E. purpurea aerial samples was determined using high-throughput 16S rRNA gene pyrosequencing. Bacterial isolates representing 38 different taxa identified to be present within E. purpurea were acquired, and the activity exhibited by the extracts of these isolates varied by over 8000-fold. Members of the Proteobacteria exhibited the highest potency for in vitro macrophage activation and were the most predominant taxa. Furthermore, the mean activity exhibited by the Echinacea extracts could be solely accounted for by the activities and prevalence of Proteobacteria members comprising the plant-associated bacterial community. The efficacy of E. purpurea material for use against respiratory infections may be determined by the Proteobacterial community composition of this plant, since ingestion of bacteria (probiotics) is reported to have a protective effect against this health condition.

The endophytic fungal community associated with the native cactus Opuntia humifusa in the United States was investigated and its potential for providing antifungal compounds. A hundred-eight endophytic fungal isolates were obtained and identified by molecular methods into 17 different taxa of the genera Alternaria, Aureobasidium, Biscogniauxia, Cladosporium, Cryptococcus, Curvularia, Diaporthe, Epicoccum, Paraconiothyrium, Pestalotiopsis and Phoma. The most frequent species associated with O. humifusa were Alternaria sp. 3, Aureobasidium pullulans and Diaporthe sp. The fungal community of O. humifusa had a high richness and diversity; additionally, the species richness obtained indicates that the sample effort was enough to recover the diversity pattern obtained. Six extracts of endophytes showed antifungal properties and (1)H NMR analyses of the extracts of Alternaria sp. 5 Ohu 8B2, Alternaria sp. 3 Ohu 30A, Cladosporium funiculosum Ohu 17C1 and Paraconiothyrium sp. Ohu 17A indicated the presence of functional groups associated with unsaturated fatty-acid olefinic protons and fatty acid methylene and methyl protons. GC-FID analysis of these extracts confirmed the presence of a mixture of different fatty acids. The (1)H NMR analyses of Biscogniauxia mediterranea Ohu 19B extracts showed the presence of aromatic compounds. From the extract of B. mediterranea we isolated the compound 5-methylmellein that displayed moderate antifungal activity against the phytopathogenic fungi Phomopsis obscurans. Our results suggest that native medicinal cacti of the United States can live symbiotically with rich and diverse endophytic communities and may be a source of bioactive molecules, including those able to inhibit or control plant disease pathogens.

A growing body of evidence suggests that endophytic bacteria within Echinacea are the main source of components responsible for enhancing innate immune function in vitro and in vivo. We have found that 97% of in vitro monocyte/macrophage activation by extracts of Echinacea and other immune enhancing botanicals is due to the bacterial components LPS and Braun type lipoproteins. Furthermore, we have found that variations in total bacterial load within Echinacea samples were highly correlated with their in vitro immune enhancing activity and content of LPS. In this study we determined the total load and community composition of the bacterial endophytes associated with aerial material of commercial and freshly harvested (wild and locally cultivated) E. purpurea plants. Over 150 endophytic bacterial isolates were cultured from fresh Echinacea samples. Extracts from these isolates varied substantially in their ability to activate macrophages in vitro. The activity of each plant extract was then compared to the sum of the activities calculated from the amount of each bacterial taxa contained in the extracted plant material. Our results indicate that the type and amount of the endophytic bacteria identified in E. purpurea aerial material accounts for 87% and 100% of the activity exhibited by the freshly harvested and commercial plant extracts, respectively.

Previous studies from our laboratory indicate that LPS and Braun type lipoproteins derived from endophytic bacteria within Echinacea are responsible for 97% of in vitro monocyte/macrophage activation by extracts of this botanical. Furthermore, we have found that changes in the levels of these two components are responsible for the variation (˜100-fold) observed in the activity of Echinacea material obtained from different commercial sources. In the current study we investigated whether agronomic factors known to influence endophyte community composition could be responsible for the wide variation in the activity that we observed in the commercially obtained plant material. Our results showed that cultivation of E. purpurea in soils containing increasing amounts of organic matter significantly enhanced the level of in vitro macrophage activation exhibited by extracts of the root material. Increasing soil organic matter content 2.5 times resulted in a 4-fold increase in macrophage activation potential exhibited by root material (p< 0.0001). Cultivation of plants in soil with different levels of moisture content did not significantly alter the level of macrophage activation exhibited by either E. purpurea root or aerial material. These results indicate that soil organic matter content is one agronomic factor that alters the amount and type of bacterial endophytes present and thereby contributes to the difference in macrophage activation potential exhibited by E. purpurea plant material.

Leishmaniasis is a chronic infectious disease caused by different Leishmania species. Global occurrences of this disease are primarily limited to tropical and subtropical regions. Treatments are available; however, patients complain of side effects. Different species of plants have been screened as a potential source of new drugs against leishmaniasis. In this study, we investigated the antileishmanial activity of cilantro (Coriandrum sativum) essential oil and its main components: (E)-2-undecenal, (E)-2-decenal, (E)-2-dodecenal, decanal, dodecanal, and tetradecanal. The essential oil of C. sativum leaves inhibits growth of Leishmani donovani promastigotes in culturewith an IC50 of 26.58 +/- 6.11 mu g/mL. The aliphatic aldehydes (E)-2-decenal (7.85 +/- 0.28 mu g/mL), (E)-2-undecenal (2.81 +/- 0.21 mu g/mL), and (E)-2-dodecenal (4.35 +/- 0.15 mu g/mL), all isolated from C. sativum essential oil, are effective inhibitors of in vitro cultures of L. donovani promastigotes. Aldehydes (E)-2-decenal, (E)-2-undecenal, and (E)-2-dodecenal were also evaluated against axenic amastigotes and IC50 values were determined to be 2.47 +/- 0.25 mu g/mL, 1.25 +/- 0.11 mu g/mL, and 4.78 +/- 1.12 mu g/mL, respectively. (E)-2-Undecenal and (E)-2-dodecenal demonstrated IC50 values of 5.65 +/- 0.19 mu g/mL and 9.60 +/- 0.89 mu g/mL, respectively, against macrophage amastigotes. These cilantro compounds showed no cytotoxicity against THP-1 macrophages.

Palicourea rigida H.B.K. (Rubiaceae), a medicinal species commonly known as douradinha, has wide distribution across ecosystems in Central and South America. This species exhibits seed dormancy delaying germination until optimal conditions for seedling growth and development are in place. While dormancy ensures species survival, it also presents a technical problem for developing P. rigida's plant production program. Thus, the objective of this study was to investigate if secondary metabolites present in seeds influence the seed dormancy of P. rigida. Mature fruits were harvested from the native habitat, in the savanna region of the State of Minas Gerais during February 2009, 2010 and 2011. The content of phenolic compounds in the seed of P. rigida was measured, and the allelopathic effects were assessed using the germination of lettuces as model to detect phytotoxicity. The P. rigida seeds geminated at rates varying between 7% and 31% with a Seed Germination Index (SGI) of 0.09. Data suggest that the phenolic compounds present in the seeds may be responsible for seed dormancy.

Stevia rebaudiana Bertoni, is a commercial source of potent non-caloric and nonsynthetic sweeteners [1]. It is used worldwide and has gained attention recently by numerous multinational enterprises in the food and beverage industry [2]. Sand was used as a substrate in an experiment carried out in a greenhouse of the United States Department of Agriculture, Agricultural Research Service, in University, Mississippi. The experimental design was completely randomized with four replications. Each replication consisted of a pot with one plant. The treatments consisted of a Hogland and Arnon (1950) nutrient solution modified with doses of calcium of 0, 2, 4, 6, 8 and 12 mmol L-1. Plants were harvested 55 days after transplantation and dried to obtain dry weight of shoot. Quantitative analysis of rebaudioside has been done following a previously described method [3]. Both dry weight of shoot and rebaudioside yield were highly influenced for doses of calcium in nutrient solution. The doses of 5.6 mmol L-1 of Ca provided the highest dry weight of shoots (Fig. A) and a dose of 4.0 mmol L-1 of Ca provided a higher rebaudioside yield (Fig. B).

Roots of Zeyheriamontana, a species native to the savanna (Cerrado) region of central Brazil, produce lapachol, a naphthoquinone with anticancer activity. Lapachol is also the precursor of β-lapachone, a novel drug candidate for preventive and adjuvant cancer therapies. The leaves of Z. montana are a renewable source of ursolic acid and oleanoic acid, compounds known for their anticancer, antioxidant and antimicrobial properties. The potential prophylactic use of β-lapachone, as well as the medicinal properties of ursolic acid, highlights the importance of this study on Z.montana’s germplasm conservation. Multiple shoots were induced on Woody Plant media with supplemented 0.1 mg·L-1 of thidiazuron (TDZ). Rooting was promoted on half strength WP (Woody Plant media containing 1.0 mg·L-1 of Indolbutiric acid-IBA). Plantlet acclimatization to ex-vitro condition was done at a 70% success rate using different substrates. It was possible to store Z.montana’s elite germplasm using in vitro cultures of media containing 2% sucrose plus 4% sorbitol for six months without subcultures.

Stevia rebaundiana (Bertoni), a perennial shrub of the Asteraceae family, is one of the most important sources of non-caloric natural sweeteners. Stevia's plant extracts and glycosides have been used for several years in Paraguay and Brazil. Studies suggest that Stevia and its glycosides exert beneficial effects on human health, including anti-hyperglycemic and antihypertensive, properties [1]. The objectives of this study were to evaluate Stevia's cold hardiness in Mississippi and to determine the effects of different harvest schedules on leaf production and yield of diterpene glycosides. Plants purchased from Ritchers Herbs were the source of nodal explants for in vitro clonal propagation. Rooted plantlets adapted in trays were later transplanted in the field of the University of Mississippi Biological Field Station. Plant spacing was 30 cm between plants in a row and 60 cm between rows. Drip irrigation supplied water to the field at rate of ¼ to ½ inches per week during summer. To protect plants during the winter of 2010 a plastic in a field low tunnel was used, while in 2011 plants remained uncovered during the winter. Leaf biomass production and yield of diterpene glycosides were evaluated based on three different harvest schedules: 1) Three harvests in 60 day intervals, two leaf harvests in 90-day intervals and a single harvest after 180 days of cultivation. Results revealed that leaf production harvested once a year yielded more than the yield of multiple harvests (2 and 3 harvests per growing season). A total of 13896.37 Kg per hectare of stem and leaves were obtained in a single harvest, and the glycosides productivity obtained from a single harvest (rebaudioside A (398.80 Kg/ha) and stevioside (512.21 Kg/ha) was also greater than the yields of multiple harvests (2 and 3 harvests). Acknowledgements: The authors would like to thank the USDA Agricultural Research Service Specific Cooperative Agreement No 58 – 6408 – 02 – 1-612 for the partial financial support to the first author, We also acknowledge the Foundation Coordination for the Improvement of Higher Education Personnel, of the Ministry of Education, Brazil” (CAPES) and National Counsel of Technological and Scientific Development (CNPq), for a Doctor's Degree scholarship granted to the second author. We also would like to thank Mr. Solomon Green III, Mr. Gregory Swain and Mr. Mark Baker for their help with extraction and field work. References: [1] Jeppesen PB, Gregersen S, et al. (2000) Metabolism, 49(2): 208 – 214.

Endophytic fungi are microorganisms that live in intercellular plant tissue without causing apparent negative effects for the host. These microorganisms are living in association with medicinal plants and have received increasing attention after the discovery that the endophytic fungus Taxomyces andreanae recovered from Taxus brevifolia, produce the anticancer paclitaxel (Taxol®). In addition, recent studies have demonstrated that the endophytic fungal communities living within medicinal plant tissues produce a wide range of metabolites with different biological activities that may be useful as scaffolds for the development of new drugs. E. purpurea is the best known of the species of the genus Echinacea, used mostly to stimulate the immune system. The aim of the present study was to determine whether any of the endophytic fungi associated with E. purpurea produced metabolites with antifungal activity. Fragments of healthy leaves and upright flower stalks of 42 plants of E. purpurea were subjected to surface sterilization and inoculated on potato dextrose agar. A total of 246 endophytic fungal isolates were recovered from 410 fragments of the plant tissues (131 from leaves and 115 from upright flower stalks). Susceptibility testing against phytopathogenic Colletotrichum species was performed using dichloromethane (DCM) crude extracts of each endophytic fungus with a final concentration of 160 µg/mL. The DCM extracts from 103 (41.9%) endophytic fungi isolates displayed moderate antifungal activity against the spore germination of at least one target fungus. While eight (3.2%) extracts displayed clear zones of fungal growth inhibition in all phytopathogenic fungi tested in the direct TLC bioautography system. Our research showed the presence of endophytic fungi associated with the medicinal plant E. purpurea and that are able to produce bioactive compounds that may be useful as sources of novel biopesticides.